The present invention relates to new alkylating antitumor agents analogous to Distamycin A, to a process for their preparation, to pharmaceutical compositions containing them and to their use as therapeutic agents. Distamycin A, whose formula is reported below, 
belongs to the family of the pyrroleamidine antibiotics and it is reported to interact reversibly and selectively with DNA-AT sequences, thus interfering with both replication and transcription. See, for a reference, Nature, 203, 1064 (1964); FEBS Letters, 7 (1970) 90; Prog. Nucleic Acids Res. Mol. Biol., 15, 285 (1975).
Several analogous to distamycin are known in the art. The international patent application WO 97/28123, in the name of the applicant, describes distamycin derivatives in which the distamycin formyl group is substituted by aromatic moieties bearing alkylating groups and the amidino moiety is replaced with other basic and non-basic nitrogen containing ending groups.
It has now been found that a new class of distamycin derivatives as defined hereinunder, wherein the distamycin formyl group is substituted by benzoheterocyclic rings bearing alkylating groups and the amidino moiety is substituted by different nitrogen-containing ending-groups, shows valuable biological properties.
Therefore, the present invention provides compounds which are distamycin derivatives of formula: 
wherein:
n is 2, 3 or 4;
A is a heteroatom selected from O and S or is a group NR, wherein R is hydrogen or C1-C4 alkyl;
B is CH or N;
R1 is hydrogen or C1-C4 alkyl;
G is selected from the group consisting of: 
and xe2x80x94Cxe2x89xa1N
wherein R5, R6, R7, R8, R9, R10, R11 and R12 are, independently from each other, hydrogen or C1-C4 alkyl; T is a group of formula (II) or (III) as defined below 
wherein p is 0 or 1; R and R3 are, independently from each other, hydrogen, C1-C4 alkyl optionally substituted by one or more fluorine atoms, or C1-C4 alkoxy; R4 is C1-C4 alkyl or C1-C3 haloalkyl; X1 and X2 are halogen atoms;
or a pharmaceutically acceptable salt thereof;
provided that at least one of R5, R6 and R7 is alkyl.
The present invention includes within its scope also all the possible isomers covered by the compounds of formula (I), both separately and in admixture, as well as the metabolites and the pharmaceutically acceptable bio-precursors (otherwise known as pro-drugs) of the compounds of formula (I).
In the present description, unless otherwise specified, the term alkyl includes straight or branched alkyl, for instance C1-C4 alkyl such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl; the term C1-C4 alkoxy includes straight or branched C1-C4 alkoxy such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy and tert-butoxy.
Preferred C1-C4 alkyl or alkoxy groups are methyl, ethyl, propyl, methoxy and ethoxy groups.
The term C1-C3 haloalkyl embraces straight or branched C1-C3 alkyl substituted by one or more halogen atoms; the term halogen atom includes fluorine, chlorine, bromine and iodine.
Preferred halogen atoms are chlorine or bromine whilst preferred C1-C3 haloalkyl groups are 2-chloroethyl or 2-bromoethyl. When substituted by fluorine atoms, the C1-C4 alkyl groups are preferably C1-C4 perfluoroalkyl groups, i.e. trifluoromethyl.
Within the compounds of formula (I) wherein T is a group of formula (II) as defined above and p is 1, the carboxamido and amino groups onto phenyl ring are in ortho, meta or para position with respect to each other; preferably the carboxamido and the amino groups are in meta or para position.
Pharmaceutically acceptable salts of the compounds of formula (I) are their salts with pharmaceutically acceptable either inorganic or organic acids such as, for instance, hydrochloric, hydrobromic, sulfuric, nitric, acetic, propionic, succinic, malonic, citric, tartaric, methanesulfonic and p-toluenesulfonic acid.
A preferred class of compounds of the present invention is that wherein, in formula (I):
n is 2 or 3;
A is O, S, NH or NCH3;
R1 is hydrogen;
G is selected from: 
xe2x80x83wherein R5, R6, and R7 are, independently from each other, hydrogen or methyl; R8, R9 and R12 are hydrogen; T is a group of formula (II) as above wherein p is 0, X1 is a chlorine atom and R4 is 2-chloroethyl or T is a group of formula (III) as above wherein X2 is chlorine or bromine. Examples of specific compounds according to the present invention, especially in the form of salts, preferably with hydrochloric acid, are the following:
1) 3-[1-methyl-4[1-methyl-4[1-methyl-4[5(xcex1-bromoacrylamido)indole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propioncyanamidine;
2) 3-[1-methyl-4[1-methyl-4[1-methyl-4[1-methyl-5(xcex1-bromoacrylamido)indazole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propioncyanamidine;
3) 3-[1-methyl-4[1-methyl-4[1-methyl-4[5(xcex1-bromoacrylamido)indole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propion-N-methylamidine;
4) 3-[1-methyl-4[1-methyl-4[1-methyl-4[1-methyl-5(xcex1-bromoacrylamido)indole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propion-N-methylamidine;
5) 3-[1-methyl-4[1-methyl-4[1-methyl-4[1-methyl-5(xcex1-chloroacrylamido)indole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propion-N-methylamidine;
6) 3-[1-methyl-4[1-methyl-4[1-methyl-4[5(xcex1-bromoacrylamido)benzofurane-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propion-N-methylamidine;
7) 3-[1-methyl-4[1-methyl-4[1-methyl-4[5(xcex1-bromoacrylamido)benzothiophene-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propion-N-methylamidine;
8) 3-[1-methyl-4[1-methyl-4[1-methyl-4[5(xcex1-bromoacrylamido)indole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propion-N,Nxe2x80x2-dimethylamidine;
9) 3-[1-methyl-4[1-methyl-4[1-methyl-4[5(xcex1-chloroacrylamido)indole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propion-N,Nxe2x80x2-dimethylamidine;
10) 3-[1-methyl-4[1-methyl-4[1-methyl-4[1-methyl-5(xcex1-bromoacrylamido)indole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propion-N,Nxe2x80x2-dimethylamidine;
11) 3-[1-methyl-4[1-methyl-4[1-methyl-4[5(xcex1-bromoacrylamido)benzofurane-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propion-N,Nxe2x80x2-dimethylamidine;
12) 3-[1-methyl-4[1-methyl-4[1-methyl-4[5(xcex1-bromoacrylamido)benzothiophene-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propion-N,Nxe2x80x2-dimethylamidine;
13) 3-[1-methyl-4[1-methyl-4[1-methyl-4[1-methyl-5(xcex1-bromoacrylamido)indazole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propion-N,Nxe2x80x2-dimethylamidine;
14) 3-[1-methyl-4[1-methyl-4[1-methyl-4[1-methyl-5(xcex1-bromoacrylamido)indole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propionamidoxime;
15) 3-[1-methyl-4[1-methyl-4[1-methyl-4[5(xcex1-bromoacrylamido)indazole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propionamidoxime;
16) 3-[1-methyl-4[1-methyl-4[1-methyl-4[5(xcex1-chloroacrylamido)indole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propionamide;
17) 3-[1-methyl-4[1-methyl-4[1-methyl-4[1-methyl-5(xcex1-bromoacrylamido)indole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propionamide;
18) 3-[1-methyl-4[1-methyl-4[1-methyl-4[5(xcex1-bromoacrylamido)benzofurane-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propionamide;
19) 3-[1-methyl-4[1-methyl-4[1-methyl-4[1-methyl-5(xcex1-chloroacrylamido)indazole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propionamide;
20) 2-[1-methyl-4[1-methyl-4[1-methyl-4[5(xcex1-bromoacrylamido)benzofurane-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]ethylguanidine;
21) 2-[1-methyl-4[1-methyl-4[1-methyl-4[5(xcex1-bromoacrylamido)benzofurane-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]ethylguanidine;
22) 3-[1-methyl-4[1-methyl-4[1-methyl-4[5(xcex1-bromoacrylamido)indole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propionitrile;
23) 3-[1-methyl-4[1-methyl-4[1-methyl-4[1-methyl-5(xcex1-bromoacrylamido)indole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propionitrile;
24) 3-[1-methyl-4[1-methyl-4[1-methyl-4[5-N,N-bis(2-chloroethyl)aminobenzofurane-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propioncyanamidine;
25) 3-[1-methyl-4[1-methyl-4[1-methyl-4[5-N,N-bis(2-chloroethyl)aminoindazole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propion-N-methylamidine;
26) 3-[1-methyl-4[1-methyl-4[1-methyl-4[1-methyl-5-N,N-bis(2-chloroethyl)aminoindazole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propion-N,Nxe2x80x2-dimethylamidine;
27) 3-[1-methyl-4-[1-methyl-4-[1-methyl-4-[5-N,N-bis(2-chloroethyl)aminoindole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propionamidoxime;
28) 3-[1-methyl-4[1-methyl-4[1-methyl-4[5-N,N-bis(2-chloroethyl)aminobenzofurane-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propionamidoxime;
29) 3-[1-methyl-4[1-methyl-4[1-methyl-4[5-N,N-bis(2-chloroethyl)aminobenzothiophene-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propionamide;
30) 2-[1-methyl-4[1-methyl-4[1-methyl-4[5-N,N-bis(2-chloroethyl)aminoindole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]ethylguanidine;
31) 2-[1-methyl-4[1-methyl-4[1-methyl-4[1-methyl-5-N,N-bis(2-chloroethyl)aminoindole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]ethylguanidine;
32) 3-[1-methyl-4[1-methyl-4[1-methyl-4[5-N,N-bis(2-chloroethyl)aminoindazole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propionitrile.
A further object of the present invention is a process for preparing the compounds of formula (I), and the pharmaceutically acceptable salts thereof, which process comprises:
(a) reacting a compound of formula: 
xe2x80x83wherein n and G are as defined above; with a compound of formula: 
xe2x80x83wherein A, B, T and R1 are as defined above; is hydroxy or a suitable leaving group; to obtain a compound of formula (I) as defined above; or
(b) reacting a compound of formula: 
xe2x80x83wherein n, A, B, G and R1 are as defined above; with a compound of formula: 
wherein X1, R2, R3, R4 and Y are as defined above; or, alternatively, with a compound of formula: 
wherein X2 and Y are as defined above; to obtain a compound of formula (I) wherein T is a group of formula (II) with p equal to 1 or a group of formula (III); or
(c) reacting a compound of formula (VIII) 
xe2x80x83wherein n, A, B, R1 and T are as defined above; with succinic anhydride, so obtaining a compound of formula (I) having G equal to xe2x80x94Cxe2x89xa1N; or
(d) reacting a compound of formula (IX): 
xe2x80x83wherein n, A, B, R1, R2, R3, R4 and X1 are as defined above; with
(i) H2Nxe2x80x94(CH2)mxe2x80x94NH2, where m is 2 or 3, to obtain a compound of formula (I) wherein G is: 
(ii) H2Nxe2x80x94CH2xe2x80x94CHO to obtain a compound of formula (I) wherein G is: 
(iii) H2Nxe2x80x94CN, so obtaining a compound of formula (I) having G equal to: 
(iv) H2Nxe2x80x94OR121 wherein R12 is as defined above, so obtaining a compound of formula (I) having G equal to: 
(v) H2Nxe2x80x94NH2, so obtaining a compound of formula (I) having G equal to: 
(vi) HNR5R6, so obtaining a compound of formula (I) having G equal to: 
xe2x80x83and then optionally with H2NR7, so obtaining a compound of formula (I) having G equal to: 
xe2x80x83wherein R5, R6, and R7 are, independently from each other, hydrogen or C1-C4 alkyl;
(vii) HNR8R9, so obtaining a compound of formula (I) having G equal to: 
xe2x80x83and then with water in an alkaline medium, so obtaining a compound of formula (I) having G equal to xe2x80x94COxe2x80x94NR8R9, wherein R8 and R9 are, independently from each other, hydrogen or C1-C4 alkyl; or
(viii) water in an alkaline medium, so obtaining a compound of formula (I) having G equal to xe2x80x94CONH2;
xe2x80x83and, if desired,
(e) converting a compound of formula (I) into a pharmaceutically acceptable salt thereof.
In the compounds of formula (V), (VIIa) and (VIIb), Y is hydroxy or a suitable leaving group such as, for instance, chloro, 2,4,5-trichlorophenoxy, 2,4-dinitrophenoxy, succinimido-N-oxy, imidazolyl group, and the like.
The condensation reactions as set forth above under processes (a) and (b) can be carried out according to known methods, for instance those described in EP-A-246,868 and in the aforementioned WO 97/28123.
The reaction of a compound of formula (IV) with a compound of formula (V) wherein Y is hydroxy is preferably carried out with a molar ratio (IV):(V) of from 1:1 to 1:2, in an organic solvent such as, e.g., dimethylsulphoxide, dimethylacetamide, dimethylformamide, ethanol, benzene, or pyridine, in the presence of an organic or inorganic base such as, e.g., triethylamine, N,Nxe2x80x2-diisopropylethylamine, or sodium or potassium carbonate or bicarbonate, and a condensing agent such as, e.g., N-ethyl-Nxe2x80x2-(3-dimethyl-aminopropyl)carbodiimide, N,Nxe2x80x2-dicyclohexylcarbodiimide, or 1-hydroxybenzotriazole hydrate. The reaction temperature may vary from about xe2x88x9210xc2x0 C. to about 100xc2x0 C., and the reaction time from about 1 to about 24 hours.
The reaction between a compound of formula (IV) and a compound of formula (V) wherein Y is a leaving group as defined above, may be carried out with a molar ratio (IV):(V) of from about 1:1 to about 1:2, in an organic solvent such as, e.g., dimethylformamide, dioxane, pyridine, tetrahydrofuran, or mixtures thereof with water, optionally in the presence of an organic base, e.g. N,Nxe2x80x2-diisopropylethylamine, triethylamine, or an inorganic base, e.g. sodium or potassium bicarbonate, at a temperature of from about 0xc2x0 C. to about 100xc2x0 C., and for a time varying from about 2 hours to about 48 hours.
The compounds of formula (IV) are known compounds, or may be prepared by known methods, for instance as described in WO 97/28123.
The compounds of formula (V) wherein Y is hydroxy and T is a group of formula (II) with p equal to 1, or a group of formula (III), can be prepared by reacting an amino compound of formula: 
wherein A, B and R1 are as defined above, with a compound of formula (VIIa) or (VIIb) as defined above.
The compounds of formula (V) wherein Y is hydroxy and T is a group of formula (II) with p equal to 0, can be prepared by reacting a compound of formula: 
wherein A, B, R1, and R4 are as defined above, with ethylene oxide and then with a halogenating agent. Before carrying out the reaction, the carboxyl group is preferably protected with a suitable protecting group according to known techniques.
The compounds of formula (V) wherein Y is a leaving group can be prepared starting from the corresponding acids through well known reactions.
The compounds of formula (X) and (XI) are commercial products, or can be obtained by known methods. See, for a reference, J. Am. Chem. Soc. 80, 4621 (1958); Helv. Chim. Acta 31, 75 (1948); Synth. Commun. 21, 959 (1991); Anti-cancer Drug Design 10, 25 (1995); J. Org. Chem. 26, 4996-97 (1961); or Synth. Commun. 24, 3129-3134 (1994).
The carboxylic acids of formula (VIIa) and (VIIb), or the derivatives thereof, are commercially available products, or may be prepared through reactions well known in organic chemistry. See, for a reference, Tetrahedron Letters 31 1299 (1990); Anti-cancer Drug Design 9, 511 (1994); JACS 62 3495 (1940); J.Org. Chem. 26 4996-97 (1961); or Synth. Commun. 24 3129-3134 (1994).
The compounds of formula (VI) can be obtained by nitro-group reduction, according to known methods, of the compounds of formula: 
wherein n, A, B, R1 and G are as defined above.
In their turn, the nitro-derivatives of formula (XII) can be obtained by reacting a compound of formula (IV) as defined above with a compound of formula: 
wherein A, B, R1 and Y are as defined above.
The compounds of formula (XIII) are known compounds, or may be obtained by known methods. See, for a reference, Tetrahedron Letters 31, 1299 (1990); Anti-cancer Drug Design 9, 511 (1994); JACS 62, 3495 (1940); J. Org. Chem. 26, 4996-97 (1963); or Synth. Commun. 24, 3129-3134 (1994). The reaction according to process (c) can be carried out analogously to what described in U.S. Pat. No. 4,738,980. The halogenating agent may be, e.g., an elemental halide, such as chlorine or bromine, or a thionyl halide, such as thionylchloride.
The reaction of a compound of formula (VIII) with succinic anhydride is preferably carried out with a molar ratio (VIII):succinic anhydride of from 1:1 to 1:3 in an organic solvent such as, e.g., dimethylsulphoxide, in the presence of an organic or inorganic base such as, for instance, diisopropylethylamine, triethylamine, sodium or potassium carbonate and the like.
The reaction temperature may vary from about 25xc2x0 C. to about 100xc2x0 C., and for a time varying from about 1 hour to about 12 hours.
The compounds of formula (VIII) are known compounds or can be prepared from known compounds through well known reactions in organic chemistry as described, for instance, in J. Med. Chem. 9, 882, (1996); J. Med. Chem. 25, 178, (1982); J. Org. Chem. 26, 4996, (1961); J. Heterocyclic Chem. 32, 1063, (1995); or Synth. Commun. 24, 3129-3134, (1994).
The reaction between a compound of formula (IX) and one of the reactants as described in points (i-vi) according to process (d), can be carried out according to known methods, for instance those described in U.S. Pat. No. 4,766,142; Chem. revs. (1961), 155; J. Med. Chem. (1984), 27, 849-857; Chem. Revs. (1970), 151; and xe2x80x9cThe Chemistry of amidines and imidatesxe2x80x9d, edited by S. Patai, John Wiley and Sons, N.Y. (1994).
The reaction in water in an alkaline medium as set forth in points (vii-viii) may be carried out according to known methods usually employed for alkaline hydrolysis, e.g. by treating the substrate with an excess of sodium or potassium hydroxide dissolved in water or into a water/organic solvent admixture, e.g. dioxane, tetrahydrofuran or acetonitrile at a temperature of from 50xc2x0 C. to about 100xc2x0 C., for a time varying from about 2 hours to about 48 hours.
In view of what above reported, it is clear to the man skilled in the art that when preparing the compounds of formula (I) according to processes (a)-(d) as set forth above, optional amino groups, i.e. R10 and/or R11 of the compounds of formula (IV) and (VI) equal to hydrogen, need to be properly protected according to conventional techniques, so as to avoid unwanted side reactions.
Likewise, the conversion of the said protected amino groups into the free amines may be carried out according to known procedures. See, for a general reference, J. Org. Chem. 43, 2285, (1978); J. Org. Chem. 44, 811 (1979); J. Am. Chem. Soc. 78, 1359 (1956); Ber. 65, 1192 (1932); and J. Am Chem. Soc. 80, 1154, (1958).
Salification of a compound of formula (I), as well as preparation of a free compound starting from a salt, may be carried out by known standard methods. Well known procedures such as, e.g., fractional crystallisation or chromatography, may also be followed for separating a mixture of isomers of formula (I) into the single isomers.
The compounds of formula (I) may be purified by conventional techniques such as, e.g., silica gel or alumina column chromatography, and/or by recrystallisation from an organic solvent such as, e.g., a lower aliphatic alcohol, e.g. methyl, ethyl or isopropyl alcohol, or dimethylformamide.
The compounds of formula (I) according to the present invention are useful as antineoplastic agents. Particularly, they show cytostatic properties towards tumor cells, so that they can be useful to inhibit growth of various tumors in mammals, including humans, such as, for instance, carcinomas, e.g. mammary carcinoma, lung carcinoma, bladder carcinoma, colon carcinoma, ovary and endometrial tumors. Other neoplasias in which the compounds of the present invention can find application are, for instance, sarcomas, e.g. soft tissue and bone sarcomas, and the hematological malignancies such as, e.g. leukemias.
The in vitro antitumor activity of the compounds of formula (I) was evaluated by cytotoxicity studies carried out on murine L1210 leukemia cells. Cells were derived from in vivo tumors and established in cell culture. Cells were used until the tenth passage. Cytotoxicity was determined by counting surviving cells after 48 hours treatment.
The percentage of cell growth in the treated cultures was compared with that of controls. IC50 values (concentration inhibiting 50% of the cellular growth in respect to controls) were calculated on dose-response.
The compounds of the invention were tested also in vivo on L1210 murine leukemia and on murine reticulosarcoma M 5076, showing a very good antitumoral activity, with the following procedure.
L1210 murine leukemia was maintained in vivo by i.v. serial transplantation. For experiments, 105 cells were injected i.p. in CD2F1 female mice, obtained from Charles River Italy. Animals were 8 to 10 weeks old at the beginning of the experiments. Compounds were administered i.v. at day +1 after tumor cells injections.
M5076 reticulosarcoma was maintained in vivo by i.m. serial transplantation. For experiments, 5xc3x97105 cells were injected i.m. in C57B16 female mice, obtained from Charles River Italy. Animals were 8 to 10 weeks old at the beginning of the experiments. Compounds were administered i.v. at day 3, 7 and 11 after tumor injection.
Survival time of mice and tumor growth were calculated and activity was expressed in term of T/C% and T.I.%.       T    /    C    =                    median        ⁢                  xe2x80x83                ⁢        survival        ⁢                  xe2x80x83                ⁢        time        ⁢                  xe2x80x83                ⁢        treated        ⁢                  xe2x80x83                ⁢        group                    median        ⁢                  xe2x80x83                ⁢        survival        ⁢                  xe2x80x83                ⁢        time        ⁢                  xe2x80x83                ⁢        untreated        ⁢                  xe2x80x83                ⁢        group              xc3x97    100  
T.I.=% inhibition of tumor growth respect to control Tox=number of mice which died for toxicity.
Tox determination was made when mice died before the control and/or tested significant body weight loss and/or spleen and/or liver size reduction were observed.
The compounds of the invention can be administered to mammals, including humans, through the usual routes, for example, parenterally, e.g. by intravenous injection or infusion, intramuscularly, subcutaneously, topically or orally. The dosage depends on age, weight and conditions of the patient and on the administration route. For example, a suitable dosage for administration to adult humans may range from about 0.1 to about 150-200 mg pro dose 1-4 times a day.
Further object of the present invention are pharmaceutical compositions, which comprise a compound of formula (I) as an active principle, in association with one or more pharmaceutically acceptable carrier and/or diluent.
The pharmaceutical compositions of the present invention are usually prepared following conventional methods and are administered in a pharmaceutically suitable form. For instance, solutions for intravenous injection or infusion may contain as a carrier, for example, sterile water or preferably, they may be in the form of sterile aqueous isotonic saline solutions.
Suspensions or solutions for intramuscular injections may contain, together with the active compound a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
In the forms for topical application, e.g. creams, lotions or pastes for use in dermatological treatment, the active ingredient may be mixed with conventional oleaginous or emulsifying excipients.
The solid oral forms, e.g. tablets and capsules, may contain, together with the active compound, diluents, e.g., lactose, dextrose, saccharose, cellulose, corn starch and potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents, e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethyl cellulose, polyvinyl-pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates, sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, for instance, lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulation. Said pharmaceutical preparations may be manufactured by known techniques, for example by means of mixing, granulating, tabletting, sugar-coating or film-coating processes.
Further object of the present invention are,the compounds of formula (I) for use in a method for treating the human or animal body by therapy.
Furthermore, the present invention provides a method for treating tumors in a patient in need of it, which comprises administering to said patient a composition of the invention.
A further object of the present invention is a combined method for treating cancer or for ameliorating the conditions of mammals, including humans, suffering from cancer, said method comprising administering a compound of formula (I), or a pharmaceutically acceptable salt thereof, and an additional antitumor agent, close enough in time and in amounts sufficient to produce a therapeutically useful effect.
The present invention also provides products containing a compound of formula (I), or a pharmaceutically acceptable salt thereof, and an additional antitumour agent as a combined preparation for simultaneous, separate or sequential use in anti-cancer therapy.
The term xe2x80x9cantitumor agentxe2x80x9d is meant to comprise both a single antitumor drug and xe2x80x9ccocktailsxe2x80x9d i.e. a mixture of such drugs, according to the clinical practice. Examples of antitumor agents that can be formulated with a compound of formula (I), or alternatively, can be administered in a combined method of treatment, include doxorubicin, daunomycin, epirubicin, idarubicin, etoposide, fluoro-uracil, melphalan, cyclophosphamide, 4-demethoxy daunorubicin, bleomycin, vinblastin, and mitomycin, or mixtures thereof.